Heterochromatin is a critical component of higher eukaryotic genomes and plays a role in chromosome segregation, recombination, and gene expression. Constitutive heterochromatin in Drosophila is characterized by the presence of transposable elements, hypoacetylated histone tails, methylated histone H3 at lysine 9, and association of HP1. The RNAi-machinery and small RNAs that correspond to repetitious elements also play a role in forming heterochromatin. Our overall goal is to dissect the process of heterochromatin formation by investigating components of the pathway required for targeting/initiation, maintenance, and spreading. To this end, we will examine the relative abundance of repeat associated small RNAs and determine the impact of mutations in the RNAi-machinery and heterochromatin components on these RNAs. Furthermore, we will test the capacity of an animal genome to target heterochromatin formation in trans through expression of a hairpin RNA corresponding to the target regions. If successful, this assay will allow us to dissect components involved in initiation and maintenance versus spreading and will be used to screen for mutations that impact heterochromatin formation. An assay for disruption of heterochromatic marks in tissue culture cells will also be developed, which could be used as an alternative screen for novel components of the heterochromatin formation pathway. [unreadable] [unreadable] [unreadable]